Abstract 1948 Complex formation of Arabidopsis thaliana alpha-amylase 3 with beta-amylase 9

Starch is a branched glucose polymer synthesized and stored in plastids by many plants as an energy reserve. This polysaccharide is degraded by a suite of enzymes, including the hydrolase beta-amylase. In Arabidopsis thaliana, there are nine beta-amylase genes, only five of which are catalytically active. One of the catalytically inactive forms in plastids is beta-amylase 9 (BAM9), which our lab has shown is expressed in response to various forms of stress. To identify the function of BAM9, we first aimed to determine the interaction partners of BAM9. Sequence alignment studies of BAM9 orthologs revealedseveral conserved surfaces suggesting potential sites for protein-protein interaction. Yeast two-hybrid assays then revealed the plastid localized alpha-amylase 3 (AMY3) as a suitable candidate for this interaction. AMY3 has been linked to stress-induced starch degradation, and our activity assays of AMY3 show enhanced activity in the presence of BAM9. BAM9 and AMY3 appear to have evolved relatively recently in the evolution of land plants. Both genes are found in flowering plant and gymnosperm genomes. Neither of the two genes is found in any non-seed plants, making it conceivable that BAM9 and AMY3 coevolved at about the same time to function as a heterodimer. To demonstrate complex formation, we cross-linked AMY3 and BAM9, then characterized the complex via small-angle X-ray scattering and SEC-MALS. Unexpectedly, we found that AMY3 alone formed a homomeric complex, which was disrupted in the presence of BAM9. These data indicate that BAM9 derepresses AMY3 self-inhibition, suggesting BAM9 is a pseudo-enzyme regulating starch degradation in response to stress. A better understanding of this mechanism could facilitate enhancing the response of plants to stress or help to improve industrial processes that rely on starch and its degradation products.